Candida tropicalis and application thereof, preparation method of tobacco flavor and tobacco flavor

Abstract

The invention relates to candida tropicalis, application of the candida tropicalis, a preparation method of tobacco flavor and the tobacco flavor, and belongs to the technical field of microorganisms. The method comprises the following steps: firstly, separating a series of saccharomycetes from a tobacco leaf sample, and screening out a saccharomycete H8 with strong fragrance from the saccharomycetes; the saccharomycetes H8 are identified by using morphology and molecular biology, the saccharomycetes H8 is candida tropicalis and is preserved in the China Center for Type Culture Collection (CCTCC), and the preservation number is CCTCC NO: M 20242185. Furthermore, the invention finds that the tobacco extract and the waste tobacco shreds are fermented by using the saccharomycetes H8, and the contents of various aroma components in the fermented fermentation liquor are obviously increased, so that the flavor of the tobacco extract is improved, the aroma amount and the aroma quality are obviously improved, and the smoke strength and the sweet aftertaste are promoted. Therefore, the candida tropicalis H8 provided by the invention has relatively great application potential in improving the quality of the tobacco products.

Description

Technical Field

[0001] The invention relates to tropical candida and application thereof, a preparation method of tobacco flavor and tobacco flavor, and belongs to the technical field of microorganisms. Background Art

[0002] In recent years, with the improvement of people's health awareness, the strengthening of tobacco control efforts and the promotion of "reducing harm by reducing tar", tobacco products must control the content of substances such as tar while pursuing quality. However, while controlling the content of substances such as tar, it is inevitable that tobacco products will have lighter smoke, less aroma, and less power, which will reduce the satisfaction of tobacco smoking and fail to better meet the physical and psychological satisfaction of tobacco smokers.

[0003] At present, there are many measures to improve the aroma quality and smoking quality of tobacco leaves, mainly including the selection and breeding of tobacco varieties, improving cultivation technology, adding flavors and fragrances to tobacco products, etc. Among them, adding flavors and fragrances to tobacco products can add pleasant aroma to tobacco products, give them unique characteristic aromas, and coordinate the aromas, so that the aromas of tobacco leaves of different types and grades are coordinated with each other, and it is easy to operate and easy to control. It is currently a more commonly used technical means to make up for the loss of aroma caused by harm reduction and tar reduction. Therefore, the quality of tobacco flavors and fragrances is crucial to improving the aroma quality and smoking quality of tobacco leaves. There are many kinds of tobacco flavors and fragrances on the market, mainly natural flavors and fragrances from tobacco and non-tobacco sources and artificial synthetic flavors. The extraction technology of natural flavors and fragrances is immature and greatly affected by natural factors, while synthetic fragrances also have single products and harsh preparation conditions. Many high-quality aroma components cannot be synthesized artificially, or the synthesis cost is too high, which limits the application of conventional flavors and fragrances.

[0004] With the rapid development of biological research, the use of microbial fermentation technology to prepare tobacco flavors and fragrances has become a research hotspot in the tobacco industry, with advantages such as not being restricted by climatic conditions, short production cycles, and being environmentally friendly. This technology is based on the richness of microbial resources and the diversity of microbial metabolites. Therefore, microorganisms with excellent characteristics are essential for the use of microbial fermentation technology to prepare tobacco flavors and fragrances.

[0005] The Chinese invention patent authorization document with an announcement date of September 3, 2021 and an announcement number of CN110669683B discloses a brewer's yeast and its use in the preparation of tobacco flavors. It specifically discloses that the preservation number of the brewer's yeast is CGMCC NO.17832. It is used to ferment tobacco extract. After analyzing the volatile aroma components of the fermentation liquid, the contents of esters, ketones, alcohols, phenols, hydrocarbons and aldehydes are significantly increased, while the contents of heterocyclic substances are reduced. The fermentation liquid has a specific aroma, is well compatible with tobacco, and can be used as a tobacco flavoring for tobacco flavoring.

[0006] The Chinese invention patent application document with the announcement date of November 29, 2022 and the publication number CN115404180A discloses a fragrant spore-producing Bacillus and its application in tobacco. Its Bacillus strain CFFSH013 is isolated from aged tobacco leaves and the preservation number is CCTCC NO: M2020635. This strain can improve the product grade of tobacco extract or tobacco extract, provide the fullness and quality of the aroma, reduce impurities, and can further oxidize carotenoid cytochromes such as lutein into aroma components such as damascenone and nicotinone in a shorter time.

[0007] The Chinese invention patent application document with the announcement date of August 6, 2024 and the publication number CN118440829A discloses a strain of aroma-producing yeast and its application in tobacco. The yeast is Saccharomycopsis amapae, isolated from fresh grape skin, with the preservation number CCTCC NO: M20232638. The yeast can effectively increase the aroma-causing components such as propylene glycol, megastigmatrienone, ethanol, damascenone, and solanone in tobacco flavorings.

[0008] Although some microorganisms that can be used for preparing tobacco flavors and fragrances by microbial fermentation technology have been screened in the prior art, the types of microorganisms obtained are relatively few, and the effect of improving tobacco leaf quality is limited. Therefore, screening microorganisms of different species that can effectively improve tobacco leaf quality is of great significance for improving the aroma quality of tobacco leaves and improving the smoking quality of cigarettes. Summary of the invention

[0009] The first object of the present invention is to provide a tropical Candida, providing a microorganism that can effectively improve the aroma quality of tobacco products.

[0010] The second object of the present invention is to provide an application of tropical Candida in improving the aroma quality of tobacco products, and to provide a simple and effective method for improving the aroma quality of tobacco products.

[0011] The third object of the present invention is to provide a method for preparing tobacco flavorings, and to provide a method for preparing tobacco flavorings that can effectively improve the aroma quality of tobacco products.

[0012] The fourth object of the present invention is to provide a tobacco flavor prepared by the method for preparing a tobacco flavor as described above, and to provide a tobacco flavor that can effectively improve the aroma quality of tobacco products.

[0013] In order to achieve the above object, a technical solution of tropical Candida in the present invention is:

[0014] A tropical Candida, the preservation number of the tropical Candida is CCTCC NO: M 20242185.

[0015] The beneficial effect of the above technical solution is that the tropical Candida of the present invention is a pioneering invention. The present invention firstly isolates a series of yeasts from tobacco leaf samples, and screens out a yeast H8 with a strong aroma; then, the yeast H8 is identified as tropical Candida by morphology and molecular biology, and is preserved in the China Center for Type Culture Collection.

[0016] Furthermore, the present invention found that the contents of various aroma components in the fermented broth, such as isobutanol, isopentanol, hydroxyacetone, 2-acetylfuran, phenylethanol, 2-acetylpyrrole, etc., were significantly increased by fermenting tobacco extract and waste tobacco shreds using yeast H8, and the above substances play a role in improving the aroma and sweetness of tobacco, which shows that yeast H8 has the function of improving the aroma quality of tobacco products. The present invention provides a new microbial resource for preparing tobacco flavors and fragrances using microbial fermentation technology, laying a foundation for improving the quality of tobacco products in the tobacco industry.

[0017] In order to achieve the above-mentioned object, the technical solution of the application of tropical Candida in improving the aroma quality of tobacco products in the present invention is:

[0018] The invention discloses an application of tropical Candida in improving the aroma quality of tobacco products, wherein the tropical Candida is used to ferment tobacco products.

[0019] The beneficial effect of the above technical scheme is that: the present invention separates a yeast H8 from tobacco leaves, which is identified as tropical Candida through morphology and molecular biology. After fermenting tobacco extract and tobacco shreds with it, the content of components such as isobutanol, isopentanol, hydroxyacetone, 2-acetylfuran, phenylethanol, 2-acetylpyrrole, etc. can be significantly increased, thereby increasing the aroma amount and aroma quality, thereby improving the sensory quality of tobacco products.

[0020] As a further improvement, the tobacco product includes tobacco extract, tobacco shreds, and tobacco leaves.

[0021] In order to achieve the above object, the technical scheme of a method for preparing tobacco flavoring in the present invention is:

[0022] A method for preparing tobacco flavoring comprises inoculating tropical candida into a culture medium containing tobacco products for fermentation, and performing solid-liquid separation to obtain the tobacco flavoring.

[0023] The beneficial effect of the above technical scheme is that: the present invention finds that the fermentation filtrate containing tobacco products is fermented by using the selected tropical Candida H8, and the components such as isobutanol, isopentanol, hydroxyacetone, 2-acetylfuran, phenylethyl alcohol, and 2-acetylpyrrole are significantly increased. Further, the present invention sprays the fermentation filtrate (i.e., tobacco flavoring) on ​​the surface of tobacco shreds, and performs sensory evaluation after processing into cigarettes. It is found that compared with the control group, the cigarettes sprayed with the tobacco flavoring prepared by the present invention have a significantly increased amount of aroma of smoke produced by smoking, a stronger fragrance, a greater irritation, an enhanced permeability of the cigarette, and a slightly improved strength and sweet aftertaste. The present invention provides a new method for preparing high-quality tobacco flavorings, and lays a foundation for supplementing the aroma of tobacco products.

[0024] As a further improvement, the tobacco product-containing culture medium includes tobacco extract, tobacco shreds, and water; the mass ratio of the tobacco extract, tobacco shreds, and water is (1-1.5):(1-1.5):10.

[0025] The beneficial effect of the above technical solution is that tobacco extract is a relatively common material component for supplementing and improving tobacco aroma and its analytical properties. Tobacco extract is generally prepared from tobacco leaf fragments in cigarette production, which can increase the original aroma of tobacco and is widely used in the cigarette industry. However, due to the different grades of tobacco leaves used and different preparation processes, many tobacco extracts still have problems such as insufficient aroma richness. The present invention uses the screened tropical Candida H8 to ferment tobacco extract and waste tobacco, and the components in the fermentation liquid that affect the aroma and sweetness of the aroma tobacco are significantly increased.

[0026] As a further improvement, the fermentation condition is 25-32° C. for 24-48 hours.

[0027] In order to achieve the above-mentioned object, the technical scheme of a tobacco flavor prepared by the method for preparing a tobacco flavor as described in the present invention is:

[0028] A tobacco flavor prepared by the method for preparing a tobacco flavor.

[0029] The beneficial effect of the above technical solution is that after the tobacco flavoring prepared by the present invention is sprayed on the surface of tobacco shreds, the aroma quality of cigarettes prepared from the tobacco shreds can be significantly improved, thereby enhancing the sensory experience of smokers. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 This is a colony morphology diagram of yeast H8 in Example 2 of the present invention;

[0031] Figure 2 The phylogenetic tree constructed based on the 26S rDNA gene sequence in Example 2 of the present invention;

[0032] Figure 3 The GC-MS test results of the aroma components in the fermentation broth in Example 3 of the present invention (in the figure, a is the CK group and b is the yeast H8 fermentation group). DETAILED DESCRIPTION

[0033] Yeast is a very important type of flora in fermented foods, which can produce a variety of flavor substances, such as esters, alcohols, etc., and plays an important role in enhancing the flavor of fermented foods. It can improve the flavor of fermented foods, and yeast is a microorganism with high biosafety. Therefore, in order to screen strains that can enhance tobacco flavor, increase tobacco aroma, and improve tobacco quality, the present invention first isolates a series of yeasts from tobacco leaf samples, and then further screens to obtain tropical Candida H8 that can effectively improve tobacco products, and deposits it in the China Center for Type Culture Collection (CCTCC) on October 14, 2024, with the deposit number of CCTCC NO: M 20242185.

[0034] The present invention is further described below in conjunction with specific implementation methods, but the protection scope of the present invention is not limited thereto; unless otherwise specified, various reagents, instruments, etc. used in the examples are commercially available products.

[0035] The formula and preparation process of the culture medium used in the following examples are as follows:

[0036] Preparation of YPD solid culture medium: prepare according to the ratio of 10 g / L yeast extract, 20 g / L peptone, 20 g / L glucose, and 15 g / L agar, and sterilize at 115°C for 30 min.

[0037] Preparation of YPD liquid culture medium: prepare according to the ratio of 10 g / L yeast extract, 20 g / L peptone, and 20 g / L glucose, and sterilize at 115°C for 30 min.

[0038] Preparation of PDA medium: Select the potato dextrose agar (PDA) medium (Cat. No. 021050) from Guangdong Huankai Microbiological Technology Co., Ltd., configure it at 40.1 g / L, and sterilize it at 115°C for 30 min.

[0039] Preparation of tobacco extract culture medium: Tobacco extract (prepared from Yunyan 87 tobacco leaves), waste tobacco and water were mixed in a mass ratio of 1:1:10 and sterilized at 115°C for 30 minutes.

[0040] Waste tobacco: made from cut tobacco leaves from Shaanxi Province-KHP-CX24L 2021.

[0041] 1. A specific embodiment of a tropical Candida species of the present invention:

[0042] The tropical Candida of the present invention is tropical Candida (Candida tropicalis) H8, the deposit number is CCTCC M 20242185; the deposit date is October 14, 2024; the deposit unit is China Center for Type Culture Collection; the deposit address is Wuhan University, No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province. The specific implementation operation is as follows:

[0043] Example 1 Isolation and screening of yeast H8

[0044] In this example, a yeast H8 with a strong aroma was isolated from a tobacco leaf sample. The specific implementation operation is as follows:

[0045] Take 25.0g tobacco leaf sample and add 225mL sterile PBS buffer. Homogenize for 3min to obtain bacterial suspension. Take the bacterial suspension and dilute it to 10 -5 About 100 μL of the diluted bacterial suspension was spread on a YPD plate and cultured at 28°C to obtain a series of yeast strains. Experienced sensory evaluation experts conducted sensory evaluation on the solid plates of yeasts with different colony morphologies and screened out the yeast with a strong aroma, namely H8.

[0046] Example 2 Identification of yeast H8

[0047] In this example, the yeast H8 isolated and obtained in Example 1 was identified by morphology and molecular biology as Candida tropicalis. The specific implementation steps are as follows:

[0048] 1. Morphological identification

[0049] The yeast strain H8 obtained by the above screening was inoculated onto a YPD solid culture medium plate and cultured at 28°C for 2 days before observing the colony morphology. Figure 1 As shown, the colonies of yeast strain H8 are white in color, smooth and round in surface, slightly shiny, and have neat edges.

[0050] 2. Molecular Biology Identification

[0051] The yeast strain H8 was sent to BGI for 26S sequencing, and the nucleotide sequence of the yeast strain H8 26S was measured as shown in SEQ ID No. 1. The sequencing results were submitted to the NCBI database for comparison and strain identification. The identification results showed that the ITS sequence of the yeast strain H8 was 99% similar to the ITS sequence of Candida tropicalis strain (D04-13 FJ432674.1), and the phylogenetic tree was constructed using MEGA 11.0 software. The phylogenetic tree is shown in Figure 2 As shown. Figure 2 It can be seen that yeast strain H8 belongs to the Candida tropicalis yeast clade.

[0052] Combined with morphological characteristics and ITS sequence analysis, the strain H8 was identified as Candida tropicalis yeast and classified as Candida tropicalis H8. It was deposited in the China Center for Type Culture Collection (CCTCC) on October 14, 2024, with the deposit number CCTCC NO: M 20242185, and the deposit address is No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province.

[0053] The present invention discloses an application of tropical Candida in improving the aroma quality of tobacco products, a preparation method of tobacco flavorings, and specific embodiments of tobacco flavorings:

[0054] Example 3 Tobacco flavoring: Tobacco products fermented by Candida tropicalis H8

[0055] In this example, the tropical Candida H8 screened in Example 1 was used to ferment a culture medium containing tobacco products. After the fermentation was completed, the fermentation supernatant after solid-liquid separation was the tobacco flavoring. Then, the prepared tobacco flavoring was analyzed using GC-MS technology. The specific implementation operation is as follows:

[0056] A single colony of tropical Candida albicans H8 screened in Example 1 was inoculated into a YPD liquid medium and cultured in a horizontal shaker at 28°C and 180 rpm for 16 h to obtain a seed solution. The concentration of yeast strain H8 in the seed solution was controlled at OD 600= 1; inoculate the seed liquid into tobacco extract medium at 1% (v / m) inoculation amount, and culture in a horizontal shaker at 28°C and 180rpm for 48h to obtain tobacco extract fermentation; centrifuge the tobacco extract fermentation at 8000rpm for 10min, collect the supernatant, and obtain tobacco extract fermentation liquid, i.e. tobacco flavor. The control group CK is the tobacco extract medium without inoculation of yeast strain H8, and the supernatant after fermentation.

[0057] GC-MS technology was used to detect the components of tobacco flavoring and control group tobacco extract culture medium. 5 mL of fermentation broth was taken in a 20 mL headspace bottle, 1 μL of 100 μg / mL naphthalene solution was added as an internal standard substance for semi-quantitative analysis, and the extraction head was inserted into the bottle and extracted with a 50 / 30 μm DVB / CAR / PDMS coated SPME fiber at 80 ° C for 30 minutes. After extraction, the extraction head was inserted into the injection port of the GC-MS system and desorbed for 5 minutes.

[0058] GC conditions: Chromatographic column: HP-INNOWAX (30m×0.25mm, 0.25μm); temperature program: initial temperature is 40℃, hold for 3min, increase to 180℃ at a rate of 10℃ / min, hold for 0min, and then increase to 260℃ at a rate of 4℃ / min; carrier gas He; flow rate 1mL / min; injection port temperature 260℃; non-split mode; desorption time 5min.

[0059] MS conditions: electron impact ion source (EI), electron energy 70 eV, ion source temperature 230 °C, quadrupole temperature 150 °C, full scan mode, scanning mass range 30-600 amu.

[0060] Test results such as Figure 3 The search results in the NIST 17.0 database are shown in Table 1.

[0061] Table 1 GC-MS detection results of aroma components in fermentation broth of Candida tropicalis H8

[0062]

[0063]

[0064] Note: “-” in Table 1 means that the ingredient was not detected.

[0065] analyze Figure 3As shown in Table 1, the contents of isobutanol, isopentanol, hydroxyacetone, 2-acetylfuran, phenylethanol, 2-acetylpyrrole and other components in tobacco extract fermentation liquid increased significantly; and these substances play a role in improving the aroma and sweetness of tobacco in tobacco, which also shows that the tobacco extract fermentation liquid formed after fermentation by yeast strain H8 can theoretically improve the aroma of tobacco products and increase the sense of salivation. Therefore, the tobacco extract fermentation liquid obtained from tobacco extract fermented by yeast strain H8 can theoretically improve the flavor of tobacco extract, increase the aroma amount and aroma quality, and thus improve the sensory quality of tobacco extract.

[0066] Example 4 Evaluation of the aroma quality of tobacco flavoring

[0067] In this example, the tobacco flavoring prepared in Example 3 was sprayed onto the surface of shredded tobacco, processed into cigarettes, and then the sensory quality of the cigarettes was evaluated. The specific implementation operations are as follows:

[0068] The tobacco extract fermentation liquid prepared in Example 3 and the control group CK were sprayed on the surface of the leaf group formula tobacco of Jilin Tobacco Industry Co., Ltd. at a ratio of 0.75% by weight of the tobacco, and then the tobacco was placed at 28°C and 68% relative humidity to balance the moisture; after the moisture balance was completed, the cigarettes were rolled and sensory evaluation was carried out. The sensory evaluation specifically refers to the evaluation results given by an 8-person evaluation team after smoking according to the tobacco industry standard YC / T 415-2011 of the People's Republic of China. Five key indicators were selected from the evaluation indicators for evaluation, and the evaluation results are shown in Table 2.

[0069] Table 2 Sensory evaluation results of cigarettes

[0070]

[0071]

[0072] It can be seen from Table 2 that compared with the tobacco extract of the control group that was not treated with the yeast strain H8, the tobacco extract fermentation liquid (the tobacco flavoring prepared by the present invention) was used to treat the tobacco shreds. The aroma of the smoke produced by smoking was significantly increased, the fragrance was more intense, the irritation was greater, the permeability of the cigarette was enhanced, and the strength and aftertaste were slightly improved; this is consistent with the component properties of the tobacco extract fermentation liquid shown in Table 1, and it also proves that the tobacco flavoring provided by the embodiment of the present invention, which is mainly prepared by fermenting the tobacco extract with the yeast strain H8, can effectively improve the flavor of the tobacco extract, and has great application potential in improving the quality of tobacco extract.

[0073] In summary, the present invention obtains a yeast strain H8 by a directional screening method, which is identified as tropical Candida tropicalis by morphology and molecular biology, and is fermented and cultured with tobacco extract and waste tobacco as culture medium. The contents of components such as isobutanol, isopentanol, hydroxyacetone, 2-acetylfuran, phenylethanol, and 2-acetylpyrrole in the fermentation supernatant are significantly increased, thereby achieving the improvement of the flavor of tobacco extract, significantly improving the aroma amount and aroma quality, and promoting the strength of smoke and sweet aftertaste. This fully shows that the tropical Candida tropicalis H8 provided by the present invention has great application potential in improving the quality of tobacco products.

[0074] Finally, it is noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A tropical Candida, characterized in that: The deposit number of the tropical Candida is CCTCC NO: M20242185.

2. A use of the tropical Candida albicans as claimed in claim 1 in improving the aroma quality of tobacco products, characterized in that: The tropical Candida is used to ferment tobacco products.

3. The use of Candida tropicalis according to claim 2 in improving the aroma quality of tobacco products, characterized in that: The tobacco products include tobacco extract, tobacco shreds and tobacco leaves.

4. A method for preparing tobacco flavoring, characterized in that: The tropical Candida albicans described in claim 1 is inoculated into a culture medium containing tobacco products for fermentation, and the tobacco flavor is obtained by solid-liquid separation.

5. The method for preparing tobacco flavoring according to claim 4, characterized in that: The tobacco product-containing culture medium comprises tobacco extract, tobacco shreds and water; the mass ratio of the tobacco extract, tobacco shreds and water is (1-1.5):(1-1.5):

10.

6. The method for preparing tobacco flavoring according to claim 4 or 5, characterized in that: The fermentation conditions are as follows: fermentation at a temperature of 25 to 32° C. for 24 to 48 hours.

7. A tobacco flavor prepared by the method for preparing a tobacco flavor according to any one of claims 4 to 6.

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